Saccharomyces boulardii is a non-pathogenic yeast that has been used safely for many decades to protect against intestinal injury, inflammation and secretion caused by a variety of bacterial enteric pathogens including Ciostridium difficile and Shigellae. The longterm goal of this project is to define specific cellular and molecular mechanisms for the protective and therapeutic effects of probiotic agents such as S. boulardii in intestinal infection and inflammation. The broad-ranging beneficial effects of S. boulardii led us to hypothesize that this probiotic yeast may act through modulation of host intestinal inflammatory response pathways. Our preliminary data demonstrate that S. boulardii produces a soluble factor that prevents IkappaBalpha degradation, NF-kappaB nuclear translocation and NF-kappaB-mediated IL-8 gene expression in human intestinal epithelial cells or monocytes. Purification and characterization studies indicate that the S. boulardii antiinflammatory factor (SAIF) is a small (approximately 1 kDa), heat stable, water soluble glycan. The Specific Aims of this proposal are: Aim 1: To determine the mechanism whereby S. boulardii and SAIF modulate NF-kappaB activation and pro-inflammatory gene expression in monocytes and intestinal epithelial cells exposed to bacterial products (C. difficile toxin A, LPS), or endogenous inflammatory mediators (IL-1betap, TNFalpha). In collaboration with project 1 we will also examine the ability of S. boulardii to modulate NF-kappaB signaling in response to the gut neuropeptides CRH and urocortin II. Aim 2: To determine whether and by which mechanism S. boulardii and SAIF prevent MAP kinase activation and signaling. This aim will also test the hypothesis that S. boulardii blocks Ras/Erk MAP kinase signaling through an inhibitory effect on EGF receptor kinase activation. Aim 3: To elucidate the mechanisms whereby S. boulardii and SAIF augment intestinal epithelial barrier function in polarized T84 monolayers and in murine small intestinal mucosal strips (in collaboration with projects 3 and 4). Aim 4: To determine whether S. boulardii and SAIF can prevent intestinal injury, inflammation and loss of barrier function in both murine and human xenograft intestinal mucosa exposed to C. difficile toxin A (in collaboration with Core B). Examination of these specific aims will advance our understanding of the cellular and in vivo mechanisms whereby a probiotic yeast and/or its soluble products can prevent and treat gastrointestinal infectious and inflammatory diseases in humans.